Project Summary: Critically short telomeres give rise to replicative senescence and apoptosis which are avoided in most cancer cells by upregulating telomerase, a reverse transcriptase that elongates telomeres. However, for effective telomere elongation, the G-quadruplex (GQ) secondary structures that form in telomeres should be unfolded. GQ structures (GQs) form in guanine-rich regions of the genome, are highly stable, and have a distinct layered structure. Stabilizing GQs in cancer cells by small molecules (SMs), which specifically bind to this layered structure, has emerged as a potentially effective method to prevent telomere elongation. Promoter regions of DNA and untranslated regions of RNA are also particularly rich in GQ-forming sequences. GQs in these regions and SMs that stabilize them have been utilized to regulate transcription or translation level gene expression. Due to their unique characteristics, GQs have also found use in biotechnological applications as sensors, switches, building blocks, and as effectors. These medical and biotechnological applications have motivated an intense effort for screening and synthesizing GQ stabilizing SMs. The efficacy of these SMs is typically evaluated by thermal melting assays which measure the additional stability provided by SMs when they bind to isolated GQs. However, in a physiological setting GQs constantly interact with destabilizing proteins and recent single molecule studies demonstrated rich dynamics during these interactions, with orders of magnitude variations in rates associated with protein-mediated GQ unfolding depending on the protein-GQ pair of interest. How SMs would influence these broadly varying dynamic interactions will ultimately determine their influence on GQ. Yet, these questions are largely unknown and unexplored since thermal melting assays on isolated GQs and SMs fail to capture these complexities. There is a need for more sophisticated assays that can provide deeper insights on the underlying mechanisms for how SMs could effectively stabilize GQs against relevant cellular agents. The goal of this proposal is to reveal the effects of SMs on GQ stability, folding kinetics, and protein-GQ interactions at the single molecule level. Our extensive work on protein-GQ interactions, particularly utilizing single molecule Frster resonance energy transfer, will provide an essential reference point to identify the influence of SMs on these interactions. The specific aims of this proposal are: (1) Revealing the influence of SMs on folding dynamics of GQ, including transition rates between intermediate folding states and GQ; (2) Revealing the influence of SMs on GQ stability and folding dynamics when interacting with physiologically significant proteins including RPA, POT1/TPP1, BLM, WRN, and RECQ5; (3) Revealing the influence of SMs on the competition between GQ formation and dsDNA formation for non-telomeric GQ. The outcomes of proposed studies will provide new insights to guide the design of more potent SMs which would function as more effective biotechnological tools and anti-cancer drugs, making them potentially significant for public health as well.